Methods and compositions to produce rice resistant to accase inhibitors

ABSTRACT

Mutant rice resistant/tolerant to ACCase inhibiting herbicides, in particular FOP herbicides, are listed in Table 1. The ACCase inhibiting herbicides used for selection include quizalofop. An exemplary mutant rice tolerant to an ACCase herbicide is disclosed, with a rice genome having G2096S or the equivalent, in the carboxyl transferase domain of the ACCase coding gene, using the Black-Grass numbering system. This mutation shows differential response to FOPs vs. DIMs herbicides, and a greater differential with comparable non-resistant rice lines. Methods to control weeds and methods to produce herbicide resistant rice including transgenic rice, are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending U.S. application Ser.No. 13/554,675, filed Jul. 20, 2012, which claims priority from U.S.Provisional Application No. 61/510,585, filed Jul. 22, 2011, and U.S.Provisional Application No. 61/541,832, filed Sep. 30, 2011. Thedisclosures set forth in the referenced applications are incorporatedherein by reference in their entireties.

BACKGROUND

Novel rice plants are described and disclosed that are characterized bytolerance/resistance to herbicides that are ACCase inhibitors andexhibit other characteristics beneficial to rice crops. Methods tocontrol weeds by use of herbicide resistant rice in fields, and methodsto produce herbicide resistant rice using e.g. transgenes encoding for amutant ACCase enzyme, are also disclosed. The disclosures set forth inthe referenced applications are incorporated herein by reference intheir entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 20, 2012, isnamed 119566_SEQ_ST25.txt and is 32,768 bytes in size.

Value of Rice Crops

Rice is an ancient agricultural crop and is today one of the principalfood crops of the world. There are two cultivated species of rice: Oryzasativa L., the Asian rice, and Oryza glaberrima Steud., the Africanrice. The Asian species constitutes virtually all of the world'scultivated rice and is the species grown in the United States. Threemajor rice producing regions exist in the United States: the MississippiDelta (Arkansas, Mississippi, northeast Louisiana, southeast Missouri),the Gulf Coast (southwest Louisiana, southeast Texas), and the CentralValleys of California.

Rice is one of the few crops that can be grown in a shallow flood as ithas a unique structure allowing gas exchange through the stems betweenthe roots and the atmosphere. Growth in a shallow flood results in thebest yields and is the reason that rice is usually gown in heavy claysoils or soils with an impermeable hard pan layer just below the soilsurface. These soil types are usually either not suitable for othercrops or at the best the crops yield poorly.

The constant improvement of rice is imperative to provide necessarynutrition for a growing world population. A large portion of the worldpopulation consumes rice as their primary source of nutrition. Riceimprovement is carried out through conventional breeding practices andby recombinant genetic techniques. Though appearing straight forward tothose outside this discipline, crop improvement requires keen scientificand artistic skill.

Although specific breeding objectives vary somewhat in the differentrice producing regions, increasing yield is a primary objective in allprograms.

Plant breeding begins with the analysis and definition of strengths andweaknesses of the current cultivars, followed by the establishment ofprogram goals, to address the latter including the definition ofspecific breeding objectives. The goal is to combine in a singlecultivar an improved combination of desirable traits from the parentalsources. These important traits may include higher yield, resistance toenvironmental stress, diseases and insects, better stems and roots,tolerance to low temperatures, better agronomic characteristics, andgrain quality.

The breeder initially selects and crosses two or more parental lines,followed by selection among the many new genetic combinations. Thebreeder can theoretically generate billions of new and different geneticcombinations via crossing. The breeder has no direct control at thecellular level; therefore, two breeders will never develop the sameline, or even very similar lines, having the same rice traits.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops such as rice. Two parents which possessfavorable, complementary traits are crossed to produce an F₁ generation.One or both parents may themselves represent an F₁ from a previouscross. Subsequently a segregating population is produced, growing theseeds resulting from selfing one or several F₁s if the two parents arepure lines, or by directly growing the seed resulting from the initialcross if at least one of the parents is an F₁. Selection of the bestindividuals may begin in the first segregating population or F₂; then,beginning in the F₃, the best individuals in the best families areselected. “Best” is defined according to the goals of a particularbreeding program e.g., to increase yield, resist diseases. Overall amultifactorial approach is used to define “best” because of geneticinteractions. A desirable gene in one genetic background may differ in adifferent background. In addition, introduction of the gene may disruptother favorable genetic characteristics. Replicated testing of familiescan begin in the F₄ generation to improve the effectiveness of selectionfor traits with low heritability. At an advanced stage of inbreeding(i.e., F₆ and F₇), the best lines or mixtures of phenotypically similarlines are tested for potential release as new parental lines.

Backcross breeding has been used to transfer genes for a highlyheritable trait into a desirable homozygous cultivar or inbred linewhich is the recurrent parent. The source of the trait to be transferredis called the donor parent. The resulting plant is expected to have theattributes of the recurrent parent (e.g., cultivar) and the desirabletrait transferred from the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theprocess is used to recover all of the beneficial characteristics of therecurrent parent with the addition of the new trait provided by thedonor parent.

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for at least three or more years. The best lines arecandidates for new commercial varieties or parents of hybrids; thosestill deficient in a few traits may be used as parents to produce newpopulations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from 8 to 12 years from the time the firstcross is made and may rely on the development of improved breeding linesas precursors. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

The improvement of rice through breeding is restricted to the naturalgenetic variation in rice and hybridizing species, such as wild rice.The introduction of new variation in a breeding program is usuallythrough the crossing program as described, such as pedigree or backcrossbreeding. However, occasionally natural mutations are found that resultin the introduction of new traits such as disease resistance or heightchanges. Breeders have also developed new traits by inducing mutations(small changes in the DNA sequence) into a rice genome. Commonly, EMS orsodium azide plus MNU are used as mutagenic agents. These chemicalsrandomly induce single base changes in DNA, usually of G and C changedto A and T. Most of these changes have no effect on the crop as theyfall either outside the gene coding regions or don't change the aminoacid sequence of the gene product.

The breeder has no direct control of mutation sites in the DNA sequence.The identification of useful changes is due to the random possibilitythat an effective mutation will be induced and that the breeder will beable to select that mutation. Seeds are treated with the mutagenicchemical and immediately planted to grow and produce M2 seed. The M2seed will carry numerous new variations; therefore, no two experimentswill produce the same combinations. Among these variations new traitspreviously not existing in rice and unavailable for selection by a plantbreeder may be found and used for rice improvement.

To find new traits the breeder must use efficient and strategicselection strategies as the process is completely random and has anextremely low frequency of useful new combinations. Among thousands ofinduced new genetic variants there may be only one with a desirable newtrait. An optimal selection system will screen through thousands of newvariants and allow detection of a few or even a single plant that mightcarry a new trait. After identifying or finding a possible new trait thebreeder must develop a new cultivar by pedigree or backcross breedingand extensive testing to verify the new trait and cultivar exhibitsstable and heritable value to rice producers.

Using recombinant genetic techniques, nucleic acid molecules withmutations, that encode improved characteristics in rice, may beintroduced into rice with commercially suitable genomes. After amutation is identified, it may be transferred into rice by recombinanttechniques.

Applications of Herbicide Resistance Patents in Rice

Weeds in crops compete for resources and greatly reduce the yield andquality of the crop. Weeds have been controlled in crops through theapplication of selective herbicides that kill the weeds but do not harmthe crop. Usually selectivity of the herbicides is based on biochemicalvariations or differences between the crop and the weeds. Someherbicides are non-selective, meaning they kill all or almost allplants. Non-selective or broad spectrum herbicides can be used in cropsif new genes are inserted that express specific proteins that conveytolerance or resistance to the herbicide. Resistance to herbicides hasalso been achieved in crops through genetic mutations that alterproteins and biochemical processes. These mutations may arise in nature,but mostly they have been induced in crops or in vitro in tissuecultures. Unfortunately in some instances, especially with repeated useof a particular herbicide, weeds have developed resistance through theunintended selection of natural mutations that provide resistance. Whenweeds become resistant to a particular herbicide, that herbicide is nolonger useful for weed control. The development of resistance in weedsis best delayed through alternating the use of different modes of actionto control weeds, interrupting development of resistant weeds.

Rice production is plagued by a particularly hard to control weed calledred rice. The difficulty arises because red rice is so geneticallysimilar to cultivated rice (they occasionally cross pollinate) thatthere are no selective herbicides available that target red rice, yet donot harm the cultivated rice. Control is currently provided incommercial rice production through the development of mutations found inrice that render rice resistant to broad spectrum herbicides e.g.imidazolinone and sulfonylurea herbicides.

Finding new mutations in rice that makes it resistant to herbicides, andto combinations of herbicides with alternative modes of action wouldgreatly benefit rice production. Obtaining and incorporating genes forherbicide resistance into rice genomes with additional favorablecharacteristics and alternative resistances is challenging,unpredictable, time consuming and expensive, but necessary to meet theworld's increasing food needs.

SUMMARY

Described herein are distinctive rice lines with unique resistances toherbicides with alternative modes of action. These rice lines shouldextend the useful life of several herbicides due to being able to rotatethe kinds of herbicides applied in grower's fields thus slowing thedevelopment of weed resistance. Several methods are possible to deploythese resistances into hybrids or varieties for weed control, as well asoptions for hybrid seed production. The rice lines described hereinrepresent new methods for weed control in rice and can be deployed inany of many possible strategies to control weeds and provide forlong-term use of this and other weed control methods. In particular,mutant rice tolerant to ACCase inhibiting herbicides is disclosed. Theseare plants with defined amino acid sequences.

For example, rice with the ACCase mutant G2096S is already agronomicallyadapted and through breeding or backcrossing as described herein, willprovide herbicide resistance in commercially suitable biologicalmaterial.

A mutant rice tolerant to an ACCase inhibitor herbicide is disclosedthat has a mutation G2096S in the carboxyl transferase coding region ofthe ACCase gene, using the Black Grass (Alopecurus myosuroides)numbering system. The mutation makes the acetyl-Coenzyme A carboxylaseenzyme tolerant/resistant to ACCase inhibitors used as herbicides.

Cells derived from herbicide resistant seeds, plants grown from suchseeds and cells derived from such plants, progeny of plants grown fromsuch seed and cells derived from such progeny are within the scope ofthis disclosure. The growth of plants produced from deposited seed, andprogeny of such plants will typically be resistant/tolerant toacetyl-Coenzyme A carboxylase-inhibiting herbicides at levels ofherbicide that would normally inhibit the growth of a correspondingwild-type plant.

A method for controlling growth of weeds in vicinity to rice plants isalso within the scope of the disclosure. One example of such methods isapplying one or more herbicides to the weeds and to the rice plants atlevels of herbicide that would normally inhibit the growth of a riceplant. For example, at least one herbicide inhibits acetyl-Coenzyme Acarboxylase activity. Such methods may be practiced with any herbicidethat inhibits acetyl-Coenzyme A carboxylase activity and any resistantrice mutation, e.g., the three embodiments disclosed herein.

A method for growing herbicide-tolerant rice plants include (a) plantingresistant rice seeds; (b) allowing the rice seeds to sprout; (c)applying one or more herbicides to the rice sprouts at levels ofherbicide that would normally inhibit the growth of a rice plant. Forexample, at least one of the herbicides inhibits acetyl-Coenzyme Acarboxylase. Such methods may be practiced with any herbicide thatinhibits acetyl-Coenzyme A carboxylase activity.

Methods of producing herbicide-tolerant rice plants that may also use atransgene. One example of such a method is transforming a cell of a riceplant with a transgene, wherein the transgene encodes an acetyl-CoenzymeA carboxylase enzyme that confers tolerance in resulting rice plant toat least one herbicide selected from the group consisting ofaryloxyphenoxypropionate herbicides, cyclohexanedione herbicides,phenypyrazoline herbicides or combinations thereof. Any suitable cellmay be used in the practice of these methods, for example, the cell maybe in the form of a callus. An embodiment of a transgenic is onecomprising a mutation in a nucleic acid encoding ACCase, from G to S inposition 2096 (Black Grass numbering system).

A recombinant, mutagenized, synthetic, and/or isolated nucleic acidmolecule including a nucleotide sequence encoding a mutagenizedacetyl-Coenzyme A carboxylase of a plant rice, in which the amino acidsequence of the mutagenized acetyl-Coenzyme A carboxylase differs froman amino acid sequence of an acetyl-Coenzyme A carboxylase of thecorresponding wild-type plant, are within the scope of the disclosure.

Different mutations in the ACCase encoding gene are often associatedwith resistance to specific types of ACCase inhibiting herbicides(FOPS), (DIMS). The specificity of different mutations thus offers thepossibility of developing multiple modes of action for weed control inrice.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows surviving plants in quizalofop sprayed row 11USAG51477marked with a flag.

FIG. 2 shows quizalofop survivors following transplanting.

FIG. 3 shows a 11USAG52084-2 rice plant with seed set.

FIG. 4 shows DNA sequence for the carboxyl transferease coding region inthe ACCase coding gene; a single nucleotide change from G2096S isidentified in the mutant line ML0831265-01493 which is identified as09PM72399. FIG. 4 discloses SEQ ID NOS 2-4, respectively, in order ofappearance.

FIG. 5 shows comparison of protein sequences for the carboxyltransferase region of the ACCase gene; line with code 09PM72399 is theline ML0831265-01493; this line shows a change of a single amino acid atposition 2096, relative to Black-Grass; R0146 is the original linetreated with a mutagen to produce the mutation population. FIG. 5discloses SEQ ID NOS 5-7, respectively, in order of appearance.

FIG. 6 shows results of plants from ML0831265-01493 at differentapplication rates of quizalofop herbicide.

FIG. 7 shows results of plants from ML0831265-01493 with application ofdifferent ACCase type herbicides.

FIG. 8 is the mutant nucleotide sequence (SEQ ID NO: 1) that encodes anACCase enzyme with S instead of G at position 2096 (Black Grass numbersystem).

FIG. 9 shows a comparison of % injury after contact with FOP's ACCaseinhibiting herbicides in (a) rice plants with a G2096S mutation; and (b)rice plants without the mutation, with a published comparison for adifferent mutation.

DETAILED DESCRIPTION

Rice, Oryza sativa L., is an important and valuable field crop. Thus, acontinuing goal of rice breeders is to develop stable and high yieldingrice cultivars that are agronomically sound. Growers are constantlyexpecting increasing yields from new varieties and hybrids as a way toincrease their economic condition. In addition on a population levelincreasing yields is necessary due to expanding nutritional needs butlimited production resources. To accomplish this goal, the rice breedermust select and develop rice plants possessing required traits andsuperior yields.

Acetyl-Coenzyme A carboxylase (ACCase; EC 6.4.1.2) enzymes synthesizemalonyl-CoA as the start of the de novo fatty acid synthesis pathway inplant chloroplasts. ACCase in grass chloroplasts is a multifunctional,nuclear-genome-encoded, very large, single polypeptide, transported intothe plastid via an N-terminal transit peptide. The active form in grasschloroplasts is a homodimeric protein.

ACCase enzymes in grasses are inhibited by three classes of herbicidalactive ingredients. The two most prevalent classes arearyloxyphenoxypropanoates (“FOPs”) and cyclohexanediones (“DIMs”). Inaddition to these two classes, a third class phenylpyrazolines (“DENs”)has been described.

Certain mutations in the carboxyl transferase region of the ACCaseenzyme results in grasses becoming resistant to ACCase herbicides. Inthe weed Black-Grass at least five mutations have been described whichprovide resistance to FOP or DIM class of ACCase herbicides. Somemutations rendering ACCase enzymes resistant to these herbicides may beassociated with decreased fitness.

Mutation Population and Establishment

A mutation breeding program was initiated to develop proprietaryherbicide tolerant lines. A permanent mutant population was created byexposing approximately 10,000 seeds (estimated by the average weight ofa kernel) of three lines including P1003, R0146, and P1062 to mutagenssodium azide (AZ) and methyl-nitrosourea (MNU). The treated seeds werespace planted. Individual plants were harvested creating 8,281 mutationlines. The mutation lines have been maintained as a permanent mutantpopulation for trait screening.

Herbicide Screening

The permanent mutant population was screened with quizalofop herbicide.Applicants planted 2,735 M2 progeny rows from R0146, 3,774 M2 progenyrows from P1003 and 655 M2 progeny rows from P1062 in two replicationswith an estimated 250,000 plants total in each replication. Quizalofopwas applied with a rate of 15 oz/acre (115.59 gmai/ha) to the firstreplication 27 days after planting. Plants were at the 3-4 leaf stageand were actively growing when herbicide was applied. The field wasflushed the day after application. After about 9 days surviving plantswere found in four different progeny rows showing an estimated mutationrate of 0.006% (FIGS. 1-3).

Protein Sequence Comparison on Lines Showing Resistance to ACCaseHerbicides

A portion of the gene that codes for the plastidic ACCase protein wassequenced from all the plants that survived application of quizalofop.Only the carboxyl transferase coding region of the gene was sequenced(FIG. 4). All of the plants deriving from line ML0831265-01493 had amutation in the DNA sequence that caused an amino acid change in theACCase protein. One DNA base was changed in the codon for amino acid2096 relative to numbering in Black-Grass. (Gen Bank CAC84161.1, denotedas “Am”) (FIG. 5). In both rice and Black-Grass the amino acid atposition 2096 is glycine. A resistant mutation in Black-Grass changesthe amino acid at this position to alanine. The mutation found in ricesurviving quizalofop application with designation ML0831265-01493changes the DNA codon for amino acid 2096 from GGC to AGC causing aserine to be inserted in position 2096 instead of glycine. The mutantline showed resistance to quizalofop in the first screening of themutant population. Later screening with quizalofop confirmed theresistance to FOPs. The surviving mutant lines were susceptible to DIMtype ACCase inhibiting herbicides.

None of the other lines from the mutant population that survivedscreening with quizalofop carried a mutation in the carboxyl transferaseregion of the ACCase coding gene however they have been confirmed tocarry resistance to quizalofop herbicide. The resistance in these linesis likely derived from changes outside the carboxyl transferase regionof ACCase or could be derived from a different type of resistantmechanism

Resistant Plants

After herbicide treatment, the surviving plants before transplantingwere green and healthy looking whereas all surrounding plants within therow and in adjacent rows were dead. The plants after transplanting weremaintained and harvested. The progeny were maintained, tested, anddeveloped as a source of herbicide resistance in production rice (Table1). This trait is backcrossed or bred into proprietary rice lines andused to develop new varieties or hybrids that will provide producerswith an alternative mode of action to control weeds in rice. Affordingthis opportunity to growers is of great value both in providing highyields and in extending the useful life of currently used weed controltechnologies. These herbicide resistant lines can be tracked through thesimple application of herbicides to growing plants or through moleculartechniques. As the full sequence of the mutation lines is knownincluding the causal mutation for herbicide resistance, molecularmarkers can be designed, such as single nucleotide polymorphic markers,for the selection of plants and lines carrying the resistance. Thesemarkers along with herbicide bioassays facilitate the development of atleast FOP type of ACCase herbicide resistance in rice.

Selection of herbicide resistant rice in a breeding program isaccomplished by spraying the progeny material with herbicide in abioassay to observe material inheriting the resistance. Alternativelyline ML0831265-01493 may be selected by sequencing the gene regioncontaining the mutation or by creating a single nucleotide polymorphicmarker to detect the mutation.

Production of Hybrid Rice Tolerant/Resistant to ACCase Inhibitor

The practical development of the trait for weed control in rice based onthe application of ACCase FOP type of herbicides is now possible.Previously these herbicides had no application in rice because theykilled the rice plants. Any of the rice lines described is suitable tobe developed into a rice cultivar or hybrid and used in rice productionas a weed control method. The resistant trait was demonstrated to befully heritable allowing for breeding and development.

The trait was demonstrated to survive and produce normal seed set afterapplication of FOP herbicides at rates that normally kill rice. Inaddition the trait was amendable to application with multiple FOPherbicides. The level of herbicide resistance is such to allow completecontrol of red rice and other grass type weeds.

The trait is fully selectable with either an herbicide bioassay or amolecular marker allowing selection and breeding strategies to developnew rice cultivars and hybrids with FOP herbicide resistance. Theresistance provided in line ML0831265-01493 is due to a single geneacting partially dominantly or fully dominantly making it ideally suitedto be backcrossed into current commercial cultivars Alternatively theline ML0831265-01493, though lacking some key quality characteristicsfor some markets, is still agronomically suitable to be used as a parentline in a pedigree breeding program. Alternatively the line if crossedwith certain female lines may be used to directly produce hybrid seedcarrying herbicide resistance, as described herein.

Suitable lines that upon conversion with genes disclosed herein producecommercial rice resistant to ACCase inhibiting herbicides rice seedsdeposited in the ATCC as PTA-8504, PTA-8505, PTA-836 and PTA-6795.Conversion may be by breeding or recombinant methods.

EXAMPLES Example 1 Results of Quizalofop Herbicide Rate Response ofML0831265-01493 Plants

Rice lines with the G2096S mutation were tested for level of resistanceto quizalofop herbicide (Assure II®) by testing a series of differentapplication rates of the herbicide. The herbicide rate treatments wereas shown in FIG. 6. All treatments were applied at the 2-3 leaf stage.The plots were evaluated twenty-one days after application. The spraywas applied in a volume of 10 gal/acre and with 1% Crop Oil Concentrate.The treatments were evaluated as the percent injury compared to anunsprayed control plot.

The source of the G2096S mutation was line ML0831265-01493. A sample ofseed from ML0831265-01493 is deposited with the ATCC. This line is likeR0146 except that it has resistance to some ACCase herbicides due to amutation causing an amino acid change to serine instead of glycine atposition 2096 in the ACCase gene.

Four different selections of ML0831265-01493 all with the G2096Smutation were replicated three times in each treatment and tested alongwith the non-mutant R0146 line. The results are based on scoringtwenty-one days after herbicide application.

Example 2 Results of ACC Inhibitor Herbicide Rate Response of Rice Lineswith the G2096S Mutation

Rice lines (ML0831265-01493) with the G2096S mutation were tested forresponse to different ACCase inhibiting herbicides. A set of herbicides(FIG. 7) were selected and applied to three rice lines with the G2096Smutation along with the non-mutated original variety R0146. The responseof R0146 and the mutated line is shown in FIG. 7. The rate of herbicideapplication is twice the level of the labeled rate except for quizalofopand clethodim, which was twice the rate selected as a rate satisfactoryto kill rice.

All treatments were applied at the 2-3 leaf stage about 20 days afterseeds were planted. The plots were evaluated twenty-one days afterapplication. The spray was applied in a volume of 10 gal/acre and with1% Crop Oil Concentrate. The treatments were evaluated as the percentinjury compared to an unsprayed control plot.

A sample of seed from ML0831265-01493 is deposited with the ATCC. Thisline is similar to R0146 except that it has resistance to some ACCaseherbicides due to a mutation causing an amino acid change to serineinstead of glycine at position 2096 in the ACCase gene.

Three different selections of ML0831265-01493 all with the G2096Smutation were replicated two or three times in each treatment and testedalong with the non-mutant R0146 line. The results are based on scoringtwenty-one days after herbicide application.

Different mutations in the ACCase encoding gene are often associatedwith resistance to specific types of ACCase inhibiting herbicides(FOPS), (DIMS). The specificity of different mutations thus offers thepossibility of developing multiple modes of action for weed control inrice. For example, FIG. 9 shows that the G2096S mutation disclosedherein conveys greater tolerance in rice to two common FOP type ofACCase inhibiting herbicides than an alternative recently publishedmutation. The different response of rice line ML0831265-01493 to FOPherbicides shows that commercial development of this line or essentiallysimilar lines provides a new mode of action for weed control notcurrently available from other sources.

Example 3 Mutant Rice Line ML0831265-01493 Shows No Apparent Differencesfrom Non-Mutated R0146 Rice in Characteristics Other than ACCaseInhibitor Resistant Rice

In research plots the mutant line ML0831265-01493 was observed side byside with the original non-mutant line R0146. No observable differenceswere identified. The plants showed the same growth pattern. The healthand robustness of the plants also appeared similar without anydetectable differences. Some characteristics where measured also showednew significant differences between the mutant line ML0831265-01493 andthe unmutated line R0146 (Table 2). The G2096S mutation in lineML0831265-01493 shows no negative effects on the normal growth orfitness of the plants.

Example 4 Development of DNA Markers to Select Plants with the Mutation

DNA markers allow selection for certain traits without having to observethe phenotype. In the instance of line ML0831265-01493 the mutationcausing the resistance is known including the specific DNA sequence andsurrounding sequence. Knowing the sequence allows the design, making,and use of any marker system that will detect single nucleotidepolymorphisms.

In most single nucleotide detection assays a primer is labeled with aspecific fluorescent dye and synthesized based on the DNA sequence toanneal with one nucleotide at the mutation site. A second primer is alsomade carrying a second fluorescent dye of a different color to annealwith the alternative nucleotide at the mutation site. Both primers arethen allowed to anneal with a sample of DNA from an individual plant.After washing only the primers which have an annealing match remain inthe sample. The fluorescence is measured and based on the color detectedthe nucleotide at the mutation site is determined. A single colorindicates the sample was homozygous for either the non-mutant type orthe mutant type, depending on the color detected, and detection of bothcolors indicates the sample was heterozygous.

Applying single nucleotide markers for the mutation allows selection forherbicide resistance without having to observe effects of herbicideapplication on the plants. Testing by either molecular marker orphenotyping is required until a new line is proven to be homozygous forthe mutation. Molecular markers show if a line or plant is homozygous orheterozygous allowing detection of homozygosity one generation earlierthan is possible with observing the effect of applying herbicides.

Example 5 Integration of ACCase Resistance into Commercial Lines

Plants are grown from line ML0831265-01493 with the G2096S mutation(donor parent) and plants from the recurrent parent, which in thisexample is P1233. The P1233 plants are emasculated following standardcrossing procedures and pollinated with pollen from plants of lineML0831265-01493. 2-4 inches of leaf material of individual plants arecollectively used to make the crosses and to analyze the plants withmolecular markers to identify a set of polymorphic markers. It is bestto identify approximately 100 polymorphic markers evenly spaced acrossthe rice genome. Harvest F1 seed from the P1233 plants, which was usedas the female parent in the cross.

F1 seeds and the recurrent parent line, P1233 are planted and grownagain. The F1 seedlings are sprayed with herbicide to verify successfulcrossing occurred from the herbicide resistant donor parent. Thoseplants not inheriting resistance will die. In addition the plants couldalso be tested with a few of the polymorphic markers to verify they weretrue F1 plants, i.e. received markers from both parents, the crossingprocess is repeated by using the resistant F1 plants as the male orpollen donor parent to plants from line P1233 emasculated and used asthe female parent. After seeds mature the BC1F1 seed is harvested.

The BC1F1 seeds and the recurrent parent line, P1233 are planted andgrown. The BC1F1 seedlings are sprayed with herbicide to identify thosethat inherited the herbicide tolerance mutation from the donor parent.Leaf tissue is collected from tolerant seedlings and submitted to thelab for analysis with polymorphic markers. Five plants are selected thatshow the highest portion of the line P1233 genome based on the markeranalysis. They are used as a pollen donor onto emasculated plants ofline P1233. After seeds mature, the BC2F1 seed is harvested.

The BC2F1 and the recurrent parent line, P1233 are planted and grown.The BC2F1 seedlings are sprayed with herbicide to identify those thatinherited the herbicide tolerance mutation from the donor parent. Leaftissue is collected from the tolerant seedlings and submitted to the labfor analysis with all unfixed (segregating) markers. Five plants areselected that show more than 95% recovery of the P1233 genome and theseare allowed to self pollinate. The backcrossing step is repeated if noindividuals show at least 95% recovery of the P1233 genome. BC2F2 seedis harvested from individual self pollinating plants.

At least 24 individual BC2F2 seeds from each plant are planted andgrown. Leaf tissue is collected; DNA is extracted and sequenced toidentify individuals that carry the G2096S mutation in homozygouscondition. The plants are allowed to self pollinate. The BC2F3 seed fromthese plants is harvested and identified as a new herbicide tolerantline of P1233. Lines or progeny rows are grown in head rows andselections for the best row are made to advance to hybrid crossing andyield trials.

The same process may also be followed to develop other lines that carryresistance to ACCase FOP type herbicides. The recurrent parent is chosenas an S-line to develop resistance in a female parent used in hybridproduction. Alternatively resistance may be developed in more than onerecurrent parent. One recurrent parent line is the male line used in ahybrid and the other is the female line used in the hybrid to make ahybrid that carried the resistance (G2096S mutation) in a homozygouscondition. Other examples of recurrent parents may be lines carryingcurrent commercial traits such as other herbicide resistances or eventransgenic traits. Other parents could be derived from other screeningsof mutant lines and selected to combine multiple traits into a singleline.

Example 6 Development of New Commercial Lines with Resistance to ACCaseType FOP Herbicides

Resistance in rice to ACCase FOP type herbicides is developed in eitherhybrid parent lines or varieties through a breeding approach. A carefulanalysis of the line ML0831265-01493 for inherent strengths and weaknessis done to identify lines that will correct the weaknesses in lineML0831265-01493. Line ML0831265-01493 carrying the herbicide resistanceas the male parent is used so that simple bioassays (spraying the plantswith the herbicides and observing those that live as individuals thatinherited the resistance) can be applied to verify successful crosses.

After selecting one or more appropriate parents a cross is first made toone selected line. Crosses with other parents could be made in latergenerations to contribute additional traits or genetic variation. Inthis example the development process will involve only one cross toP1003 to improve the weak characteristics of the mutant lineML0831265-01493. Other parents are chosen from a mutant populationcarrying resistance to an alternative herbicide allowing multipleherbicides to be used for weed control in rice production. Parents arealso selected that carry an already developed and commercializedherbicide resistance or transgenic trait.

In the first step the selected parent line P1003 is emasculated beingused as the female and considered as providing unique characteristicsand that when recombined with line ML0831265-01493 will lead towardsdevelopment of a new variety or parent line for hybrids. Pollen from themutant line carrying the G2096S mutation ML0831265-01493 is used topollinate the emasculated plants of line P1003. The F1 seed is harvestedand planted. If desired an additional cross could be made to either ofthe parent lines or to another parent for the purpose of introducingother characteristics and genetic variation.

Growing the F1 seed and applying herbicide is done to verify the crosswas successful. The surviving plants should be true F1 and are allowedto self to produce F2 seed. The F2 seed will then be planted and againsprayed to identify plants inheriting the herbicide resistance. Theplants remaining alive should be either homozygous or heterozygous forthe resistant trait. If other traits are of interest they should also beevaluated at this stage for inheritance in the F2 plants. Select amongthe F2 plants surviving the herbicide treatment and allow them to selfpollinate to produce F3 seed. Harvest F3 seed from individual plants andmaintain as an individual F3 family.

The F3 families are then planted as rows and again herbicide applied toidentify the F2 plants and F3 families which are homozygous for theherbicide resistance. Selections are made among the F3 families that arehomozygous for the herbicide resistance for other traits andcharacteristics of interest. F4 seed is harvested from the selectedrows.

The F4 seed is used directly in yield trials to develop a new variety ortest cross to select parents to produce hybrid seed for testing in yieldtrials. Selections are made among the lines in the yield trials foryield and other target traits and characteristics such as quality. TheF4 seed should also be increased to F5 at which selections for targettraits can also be made. The F5 seed should be used again in testcrosses for yield trials with hybrid seeds as well as being put directlyinto yield trials if a variety is to be developed.

After yield trials, including multi-location and replicated testing, andfull testing of the trait response, a final selection is made toidentify one or a few lines to release as a coded line. These lines arethen used for seed increase and release as either a new variety or aparent in a hybrid.

Example 7 Herbicide Resistance Deployed in Hybrids

The herbicide resistance described in line ML0831265-01493 is likelyeither dominant or partially dominant. The resistant event is deployedin a hybrid by being integrated into the male parent, the female parentor both parents. Any combination is developed for successfullycontrolling weeds in rice with an ACCase FOP type of herbicide. Throughfollowing the process of the examples above parent lines are developedto carry the ACCase herbicide resistance. These parent lines are thenused in a hybrid seed production system to produce hybrid seed carryingthe ACCase resistance to FOP type of herbicides. The seed productionprocess involves planting the female line in rows next to the malelines. The female lines are male sterile so as to prevent selfpollination. The female lines then are pollinated by the male lines andharvested to produce F1 seed. The F1 seed is hybrid seed and is plantedby growers to produce rice grain. In the situation where a variety isdeveloped, the seed is planted in isolation and then harvested to sellto growers to produce rice grain.

In preparation to produce either hybrid or variety seed from selectedlines carrying the ACCase resistance derived from ML0831265-01493 linesmust first be purified, extensively tested, and increased.

In the example of producing hybrid seed with resistance to ACCase FOPtype of herbicides the resistance may be provided in the male parent,the female parent or both parents. Providing the resistance in bothparents could be necessary to have a high enough level of resistance toprevent herbicide damage to the rice when herbicide is applied. However,a more suitable delivery mechanism in hybrids is if the resistance canbe provided in either only the female or only the male parent.

By providing the resistance in only the male parent offers a process toeliminate any female selfed seed in a growers field. With resistancebeing only provided from the male parent then when a grower appliesherbicide to his field all of the female selfed plants will besusceptible to the herbicide and thus killed. The grower's field ischemically rogued and results in a pure stand of hybrid plants.

It is also possible to provide ACCase resistance to FOP herbicidesthrough combining with other traits. For example resistance toherbicides with alternative modes of action or other traits such asinsect resistance, drought tolerance. Combining with other traits couldbe either by conventional or transgenic methods.

In one example ACCase resistance is integrated into the female parentand an alternative herbicide resistance is integrated into the maleparent. The seed is resistant to both herbicides and a grower may useeither or both herbicides for weed control. Alternatively only one typeof resistance is delivered in a single hybrid. Both systems as well asother strategies all provide growers with additional options forcontrolling weeds and will likely extend the useful life of theherbicides. In the case of deploying in only one parent or only onehybrid any red rice that develops resistance through outcrossing willonly inherit one type of resistance and will still be controllablethrough application of an herbicide with an alternative mode of action.

Example 8 Seed Production

The herbicide resistance may be used for production of hybrid seed. Asan example, if the female parent is developed with resistance to ACCaseFOP type herbicide through inheritance from line ML0831265-01493 thenthe herbicide could be used in seed production to eliminate the maleparent. By deploying into the female parent, making it resistant, thenthe herbicide is applied to the seed production field to kill the maleplants before setting seed but after pollination. In this way the maleparent is prevented from setting seed and allows seed production fieldsto be harvested as a bulk instead of only harvesting the female rows. Inaddition the purity of hybrid seed may also be verified throughdeploying the resistances in only one parent. Any selfed seed of theother parent are killed by application of the herbicide.

DEPOSIT INFORMATION

A deposit of the RiceTec, Inc. ML0831265-01493 disclosed above andrecited in the appended claims has been made with the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110. The date of deposit was May 30, 2012. All restrictions will beremoved upon granting of a patent, and the deposit is intended to meetall of the requirements of 37 C.F.R. §§1.801-1.809. The ATCC AccessionNumber is PTA-12933. The deposit will be maintained in the depositoryfor a period of thirty years, or five years after the last request, orfor the enforceable life of the patent, whichever is longer, and will bereplaced as necessary during that period.

A deposit of the RiceTec, Inc. seeds designated ML0831265-02283disclosed above and recited in the appended claims has been made withthe American Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110. The date of deposit was Mar. 19, 2013. Allrestrictions will be removed upon granting of a patent, and the depositis intended to meet all of the requirements of 37 C.F.R. §§1.801-1.809.The ATCC Accession Number is PTA-13619. The deposit will be maintainedin the depository for a period of thirty years, or five years after thelast request, or for the enforceable life of the patent, whichever islonger, and will be replaced as necessary during that period.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. Allele is any one of many alternative forms of a gene, all ofwhich generally relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing. Process of crossing a hybrid progeny to one of theparents, for example, a first generation hybrid F1 with one of theparental genotypes of the F1 hybrid.

Blend. Physically mixing rice seeds of a rice hybrid with seeds of one,two, three, four or more of another rice hybrid, rice variety or riceinbred to produce a crop containing the characteristics of all of therice seeds and plants in this blend.

Cell. Cell as used herein includes a plant cell, whether isolated, intissue culture or incorporated in a plant or plant part.

Cultivar. Variety or strain persisting under cultivation.

Embryo. The embryo is the small plant contained within a mature seed.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics of the hybrid or cultivar, except for thecharacteristics derived from the converted gene.

Grain Yield. Weight of grain harvested from a given area. Grain yieldcould also be determined indirectly by multiplying the number ofpanicles per area, by the number of grains per panicle, and by grainweight.

Locus. A locus is a position on a chromosome occupied by a DNA sequence;it confers one or more traits such as, for example, male sterility,herbicide tolerance, insect resistance, disease resistance, waxy starch,modified fatty acid metabolism, modified phytic acid metabolism,modified carbohydrate metabolism and modified protein metabolism. Thetrait may be, for example, conferred by a naturally occurring geneintroduced into the genome of the variety by backcrossing, a natural orinduced mutation, or a transgene introduced through genetictransformation techniques. A locus may comprise one or more allelesintegrated at a single chromosomal location.

Plant. As used herein, the term “plant” includes reference to animmature or mature whole plant, including a plant from which seed orgrain or anthers have been removed. Seed or embryo that will produce theplant is also considered to be the plant.

Plant Part. As used herein, the term “plant part” (or a rice plant, or apart thereof) includes protoplasts, leaves, stems, roots, root tips,anthers, seed, grain, embryo, pollen, ovules, cotyledon, hypocotyl,glumes, panicles, flower, shoot, tissue, cells, meristematic cells andthe like.

Quantitative Trait Loci (QTL). Genetic loci that controls to some degreenumerically measurable traits that are usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Single Gene Converted (Conversion). Single gene converted (conversion)includes plants developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of an inbred are recovered, whileretaining a single gene transferred into the inbred via crossing andbackcrossing. The term can also refer to the introduction of a singlegene through genetic engineering techniques known in the art.

TABLE 1 Rice lines derived from the surviving plants in the permanentmutant population confirmed to carry resistance to quizalofop herbicide.The ATCC accession number is shown for the line with the mutation in thecarboxyl transferase region of the ACCase gene and one of the otherresistant lines. The ATCC accession number is pending for the otherline. ATCC Accession Mutation in CT Designation No. domainML0831265-01493 PTA-12933 G2096S ML0831265-02283 PTA-13619 NoneML0831265-00776 pending none

TABLE 2 Comparison of the mutant line ML0831265-01493 with the originalunmutated line R0146 showing high similarity between the two lines. Daysto Thousand 50% Plant height Sheath Kernel Yield/ Line/source Head Typecm Pubescence Color Awns Weight, g plant, g R0146 87 Erect 93 PubescentGreen None 22.4 unknown 11AG52084-2 88 Erect 92 Pubescent Green None21.6 7.8 g

1. An herbicide resistant rice plant comprising a DNA sequencecomprising a plastidic carboxyl transferase domain of an ACCase codinggene which confers resistance to an ACCase inhibiting herbicide, whereinthe herbicide is selected from the aryloxyphenoxypropanoates (“FOPs”)class of ACCase inhibiting herbicides, a rice plant with a nucleotidesequence as in positions 1680-1682 of SEQ ID NO: 1 or the codonequivalent.
 2. The rice plant of claim 1 wherein thearyloryphenoryproponates herbicide is selected from the group consistingof quizalofop, clodinafop-propargyl, haloxyfop-methyl, and combinationsthereof.
 3. An herbicide resistant/tolerant rice plant derived from arepresentative sample of seed selected from the group consisting ofseeds deposited under ATCC Accession No. PTA-12933, ATCC Accession No.PTA-13619 and combinations thereof.
 4. (canceled)
 5. (canceled)
 6. Atissue culture produced from protoplasts or cells from the rice plant ofclaim 1 wherein said cells or protoplasts of the tissue culture areproduced from a plant part selected from the group consisting of leaves,pollen, embryos, cotyledon, hypocotyl, meristematic cells, roots, roottips, pistils, anthers, flowers, stems, glumes and panicles.
 7. A riceplant regenerated from the tissue culture of claim
 6. 8. A method ofproducing a herbicide resistant rice plant of claim 1, wherein themethod comprises transforming a rice plant with a transgene, wherein thetransgene confers resistance to an herbicide in thearyloxyphenoxypropanoates (“FOPs”) class of ACCase inhibitors, and thetransgene has a nucleic acid sequence that is the coding equivalent ofSEQ ID NO: 1 and wherein resistance is due to a substitution in theamino acid sequence of ACCase at position 464 of SEQ ID NO: 7,corresponding to position 2096 of the full length BlackGrass amino acidsequence.
 9. The method of producing a herbicide resistant rice of claim8, wherein the nucleic acid molecule having SEQ ID NO: 1 comprises acodon in position 1680-1682 that encodes an amino acid substitution thatmakes the ACCase enzyme resistant to inhibitors, and further comprisespromoters, terminators, selectable markers, or other domains of theACCase coding gene, necessary to encode an ACCase enzyme resistant toherbicides.
 10. The method of claim 8 wherein the mutation associatedwith resistance encodes an amino acid substitution G to S at an aminoacid positions corresponding to 2096 of the full length BlackGrassACCase amino acid sequence for ACCase.
 11. (canceled)